Rat liver ECM incorporated into electrospun polycaprolactone scaffolds as a platform for hepatocyte culture

Abstract Liver disease is expanding across the globe; however, health‐care systems still lack approved pharmaceutical treatment strategies to mitigate potential liver failures. Organ transplantation is the only treatment for liver failure and with increasing cases of liver disease, transplant programs increasingly cannot provide timely transplant availability for all patients. The development of pharmaceutical mitigation strategies is clearly necessary and methods to improve drug development processes are considered vital for this purpose. Herein, we present a methodology for incorporating whole organ decellularised rat liver ECM (rLECM) into polycaprolactone (PCL) electrospun scaffolds with the aim of producing biologically relevant liver tissue models. rLECM PCL scaffolds have been produced with 5 w/w% and 10 w/w% rLECM:PCL and were analyzed by SEM imaging, tensile mechanical analyses and FTIR spectroscopy. The hepatocellular carcinoma cell line, HepG2, was cultured upon the scaffolds for 14 days and were analyzed through cell viability assay, DNA quantification, albumin quantification, immunohistochemistry, and RT‐qPCR gene expression analysis. Results showed significant increases in proliferative activity of HepG2 on rLECM containing scaffolds alongside maintained key gene expression. This study confirms that rLECM can be utilized to modulate the bioactivity of electrospun PCL scaffolds and has the potential to produce electrospun scaffolds suitable for enhanced hepatocyte cultures and in‐vitro liver tissue models.


| INTRODUCTION
The burden of liver disease on people and health systems is increasing globally year-on-year and estimates now state that approximately 25% of the global population will be showing signs of onset or fully fledged NAFLD. [1][2][3] This puts a quarter of the world at risk of fibrosis and hepatocellular carcinoma where currently no approved pharmaceutical treatment exists for any stage of liver disease and the eventuality of liver failure brings death without transplant liver availability.
Thus, alternative strategies must be explored for the progression of drug development methodologies and other potential treatment methodologies for liver disease. Progressions in 3D cell culture methods have delivered more realistic models of liver tissues in-vitro, both structurally and functionally. 4 Culturing cells within a 3D matrix that recapitulates complex in-vivo cell-cell and cell-matrix interactions allows for more accurate modeling of in-vivo cell behaviors.
Electrospun scaffolds provide a fibrous polymeric matrix like that of the in-vivo extra-cellular matrix (ECM) upon which cells can be attached in-vitro. 5 This realistic biomechanical environment entails a simple fabrication process and research has proven its potential in the culture of hepatic cell types for in-vitro models and regenerative cell therapy purposes. [6][7][8][9][10][11] The morphology of electrospun fibers can be tailored to alter the local mechanical environment upon which cells attach and this has shown the affect the behavior and function of hepatocytes on electrospun polycaprolactone (PCL) scaffolds. 12 Moreover, many studies have shown that the local mechanics of cell attachment substrates is proven to direct differentiation of both hepatocytes and stem cells. [13][14][15][16] The bulk of many electrospun scaffolds is generally fabricated from biocompatible synthetic or natural polymers. While these materials can provide a consistent structure with cell attachment motifs, these systems lack the specific biochemical niche that is present within the in-vivo ECM.
Hepatocytes rely on a dynamic relationship with a particular set of ECM proteins which governs the function of hepatocytes in-vivo and facilitates communication between neighboring cells and the immune system. 17,18 ECM proteins such as Collagen, Laminin and Fibronectin can be incorporated into electrospun polymer scaffolds, introducing cell-matrix interactions that improve cell attachment and even direct cell function and differentiation. 8,19,20 However, this does not capture the complexity present within native ECM which is composed of hundreds of different components categorized as Collagens, ECM glycoproteins, Proteoglycans, ECM regulators, ECM affiliated proteins and secreted factors. Introducing this complexity into hepatocyte scaffolds can be achieved by stripping cells from dissected liver tissues and incorporating the decellularised liver ECM (dLECM) into scaffold structures. 21 Introduction of the complex combination of ECM proteins into liver tissue scaffolds has proven to drive altered functional responses from hepatocytes. 9 Many studies have explored the possibilities of using dLECM for the culture of hepatocytes and have shown that dLECM from caprine, murine, porcine and human sources can support hepatocytes invitro. [22][23][24][25][26][27] Removal of cells and residual cytoplasmic and nucleic materials can be achieved through different chemical and biological methods including detergent exposure, hypertonic-hypotonic processes, acids and bases and enzymatic treatments such as with trypsin and nucleases. 28 These methods can be complemented by mechanical removal of the cytoplasmic and nucleic materials such as by mechanical agitation or by perfusion methods. Whole organ perfusion methods have been established in order to retain the entire ECM structure of whole organs in view of repopulating this structure with cells and producing transplantable organs in-vitro. 29,30 Whole organ perfusion methods have been optimized for cellular material removal and ECM protein preservation in detergent-based decellularisation of renal tissues. 31,32 Thus, dLECM obtained via whole organ perfusion also presents a valuable potential resource to explore for the develop-

| Electrospinning and scaffold preparation
Electrospinning solutions were prepared with polycaprolactone Avg. MW = 80,000 (Sigma), hexafluoroisopropanol (HFIP, Manchester Organics) and rLECM. Three scaffold groups were prepared containing 0 w/w% (Control group), 5 w/w% and 10 w/w% rLECM:PCL. rLECM scaffolds were prepared by dissolving 40 mg (5 w/w%) and 80 mg (10 w/w%) of rLECM powder in 10 ml of HFIP. After a brief agitation, 0.8 g of PCL was added to the rLECM:HFIP mixture and the solution was left to fully dissolve on a roller overnight. The solutions were then loaded into a syringe and electrospun as 10 cm wide sheets onto a rotating mandrel with an IME EC-DIG Electrospinning apparatus. The parameters for electrospinning were as follows: needle diameter = 0.4 mm, flow rate = 1.5 ml/h, needle-mandrel distance = 15 cm, mandrel rotation = 250 RPM, transverse needle movement: 10 cm width at 5 cm/s. Once electrospun, 12 mm round scaffolds were punched from the sheet material and stored at 4 C until seeding with cells.
Prior to seeding the scaffolds were washed three times in 70% ethanol and lyophilised in order to sterilize the scaffolds. Post sterilization the scaffolds were submerged in PBS with 1% Anti-Anti (Gibco) to maintain sterility before seeding.

| Scanning electron microscopy
Scaffolds were imaged using a Hitachi HT4000 Plus Scanning Electron Microscope with an accelerating voltage of 15 kV using the mixed sensor mode combining back-scatter and the secondary electron sensors.

| Mechanical testing
Tensile properties of the electrospun fiber materials were assessed using an Instron 3367 tensile testing apparatus. 5 by 1 cm samples were cut from the electrospun sheets using a scalpel. The samples were clamped 1 cm at each end within the tensile testing apparatus giving a gauge length of 3 cm for each sample. Samples were extended at 15 mm/min or 50% strain/min until failure. Calculation of the Young's Modulus at different strain bands was conducted using

| Cell viability analysis
Cell viability was assessed using the cell titer blue assay (Promega) which relies on the conversion of resazurin to resorufin within the cell.
The assay was conducted on N = 5 samples per group according to the manufacturer's instructions and the fluorescence values were measured on a Modulus II Microplate reader at ex 525 nm/em 580-620 nm.

| Albumin secretion
Secreted albumin was quantified using the Bromocresol Green Assay (BCG, Sigma). Media was changed on scaffolds 24 h prior, and media from N = 5 samples per group was preserved at À80 C before measurement. The assay was conducted as per manufacturer's instruction and absorbance at 570 nm was measured on a Modulus II Microplate Reader.  with DEPC water in preparation for cDNA production, samples were stored at À80 C. cDNA preparation was conducted using the Improm-II Reverse Transcription kit (Promega) and stored at À20 C. qPCR was conducted on cDNA samples using the SYBR green dye reagent (Qiagen) with a Lightcycler 480 (Roche). Forward and reverse primer sequences used are available in the supplementary information.

| Statistical analyses
Numerical results are presented as mean ± standard deviation (SD) unless otherwise stated. All grouped data was analyzed by one-way ANOVA with Tukey's Post-Hoc testing using the MATLAB scripting software, with comparisons between groups and timepoints.

| Scaffold mechanical properties
Analysis of the tensile properties of each of the scaffold materials ( Figure 3) between 0% and 5% strain revealed the 10% rLECM group to have a significantly reduced Young's Modulus in comparison to the PCL and 5% rLECM groups by 0.96 and 0.85 MPa, respectively. Notably, upon further extension, material stiffness dropped more sharply in the PCL only scaffold in comparison to the rLECM containing groups, as a result the Youngs modulus of PCL at 10%-15% strain is 0.78 and 0.77 MPa less than the 5% rLECM and 10% rLECM groups, respectively.

| Scaffold FTIR and CN analysis
FTIR absorbance spectra were obtained for rLECM powder, PCL fibers, 5% rLECM:PCL fibers and 10% rLECM:PCL fibers. The spectra, seen in Figure 4 show the Amide I characteristic vibrational band (1642 cm À1 ) 33 present in the rLECM, 5% rLECM fibers and 10% rLECM fibers. The peak at 1642 cm À1 is absent in the absorbance spectra for PCL and the peaks for PCL are visible in the 5% rLECM fibers and 10% rLECM fibers. Table 1

| Albumin secretion
Albumin levels within the culture media, shown in Figure 8, showed an increasing trend on the PCL only scaffold over 14 days. In the T A B L E 1 Chemical analysis of the scaffold samples showing FTIR absorbance values at 1642 cm À1 (amide I content) and CN analysis results showing the percentages of carbon and nitrogen within the scaffold materials, N = 3 and results displayed as Mean ± SD   The proliferative activity shows an inverse correlation with the tensile elastic modulus of the scaffold material. The 10% rLECM scaffold shows a significant reduction in the Youngs' modulus between 0% and 5% strain which could be driving the differences seen in the proliferative activity of the HepG2. Reports in literature show that HepG2 proliferation tends to increase with increasing matrix stiffness, due to upregulation of cell cycle related proteins cyclin-D1 and β-catenin by mechanotransduction pathways. 39,40 The stiffness range of the substrates used in these experiments however did not cover the range observed between the scaffolds in our experiment. Thus, increased HepG2 proliferation upon the rLECM scaffolds cannot be conclusively attributed to the reducing tensile mechanical properties.
Discrepancies between the proliferation on the rLECM scaffolds compared with the PCL control also implies the possibility of retained functional cell attachment motifs and growth factor ligands within the scaffold material. It is known that ECM proteins can influence proliferation of HepG2 cells. 41

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.